28. Februar 2014 Pulsfeldgelelektrophorese.1 February 28, 2014 Pulsed Field Gel Electrophoresis 1 Pulsed Field Gel Electrophoresis
28. Februar 2014 Pulsfeldgelelektrophorese.2 February 28, 2014 Pulsed Field Gel Electrophoresis 2 Overview History How does it work Theory of PFGE –Parameters and their effects Realization of PFGE with the Rotaphor Comparison with the Chef-Mapper Discussion
28. Februar 2014 Pulsfeldgelelektrophorese.3 February 28, 2014 Pulsed Field Gel Electrophoresis 3 Original Design Orthogonal field alternation electrophoresis Carle &Olson NAR 1984
28. Februar 2014 Pulsfeldgelelektrophorese.4 February 28, 2014 Pulsed Field Gel Electrophoresis 4 OFAGE – Electric Field
28. Februar 2014 Pulsfeldgelelektrophorese.5 February 28, 2014 Pulsed Field Gel Electrophoresis 5 OFAGE – Electric Field
28. Februar 2014 Pulsfeldgelelektrophorese.6 February 28, 2014 Pulsed Field Gel Electrophoresis 6 OFAGE – Electric Field
28. Februar 2014 Pulsfeldgelelektrophorese.7 February 28, 2014 Pulsed Field Gel Electrophoresis 7 OFAGE – Electric Field
28. Februar 2014 Pulsfeldgelelektrophorese.8 February 28, 2014 Pulsed Field Gel Electrophoresis 8 OFAGE – Electric Field >>90°
28. Februar 2014 Pulsfeldgelelektrophorese.9 February 28, 2014 Pulsed Field Gel Electrophoresis 9 Electrode Array Zhang et al, NAR 1991 Programmable, autonomously controlled electrode array
28. Februar 2014 Pulsfeldgelelektrophorese.10 February 28, 2014 Pulsed Field Gel Electrophoresis 10 Angle Dependency Birren et al, Electrophoresis 1989 Volz, UniTübingen, 1988 RotaphorCHEF
28. Februar 2014 Pulsfeldgelelektrophorese.11 February 28, 2014 Pulsed Field Gel Electrophoresis 11 Separation Principle < 90° Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.12 February 28, 2014 Pulsed Field Gel Electrophoresis 12 Separation Principle < 90° Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.13 February 28, 2014 Pulsed Field Gel Electrophoresis 13 Separation Principle < 90° Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.14 February 28, 2014 Pulsed Field Gel Electrophoresis 14 Separation Principle < 90° Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.15 February 28, 2014 Pulsed Field Gel Electrophoresis 15 Separation Principle < 90° Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.16 February 28, 2014 Pulsed Field Gel Electrophoresis 16 Separation Principle < 90° Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.17 February 28, 2014 Pulsed Field Gel Electrophoresis 17 Separation Principle < 90° Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.18 February 28, 2014 Pulsed Field Gel Electrophoresis 18 Separation Principle < 90° Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.19 February 28, 2014 Pulsed Field Gel Electrophoresis 19 Separation Principle PFGE (>90°) Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.20 February 28, 2014 Pulsed Field Gel Electrophoresis 20 Separation Principle PFGE (>90°) Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.21 February 28, 2014 Pulsed Field Gel Electrophoresis 21 Entscheidungsschwierigkeiten What do you mean ? You are the head now ? You have been the head last time !
28. Februar 2014 Pulsfeldgelelektrophorese.22 February 28, 2014 Pulsed Field Gel Electrophoresis 22 Trennprinzip PFGE (>90°) Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.23 February 28, 2014 Pulsed Field Gel Electrophoresis 23 Trennprinzip PFGE (>90°) Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.24 February 28, 2014 Pulsed Field Gel Electrophoresis 24 Trennprinzip PFGE (>90°) Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.25 February 28, 2014 Pulsed Field Gel Electrophoresis 25 Trennprinzip PFGE (>90°) Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.26 February 28, 2014 Pulsed Field Gel Electrophoresis 26 Trennprinzip PFGE (>90°) Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.27 February 28, 2014 Pulsed Field Gel Electrophoresis 27 Trennprinzip PFGE (>90°) Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.28 February 28, 2014 Pulsed Field Gel Electrophoresis 28 Trennprinzip PFGE (>90°) Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.29 February 28, 2014 Pulsed Field Gel Electrophoresis 29 Trennprinzip PFGE (>90°) Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.30 February 28, 2014 Pulsed Field Gel Electrophoresis 30 Trennprinzip PFGE (>90°) Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.31 February 28, 2014 Pulsed Field Gel Electrophoresis 31 Trennprinzip PFGE (>90°) Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.32 February 28, 2014 Pulsed Field Gel Electrophoresis 32 Trennprinzip PFGE (>90°) Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.33 February 28, 2014 Pulsed Field Gel Electrophoresis 33 Trennprinzip PFGE (>90°) Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.34 February 28, 2014 Pulsed Field Gel Electrophoresis 34 Trennprinzip PFGE (>90°) Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.35 February 28, 2014 Pulsed Field Gel Electrophoresis 35 Trennprinzip PFGE (>90°) Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.36 February 28, 2014 Pulsed Field Gel Electrophoresis 36 Trennprinzip PFGE (>90°) Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.37 February 28, 2014 Pulsed Field Gel Electrophoresis 37 Trennprinzip PFGE (>90°) Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.38 February 28, 2014 Pulsed Field Gel Electrophoresis 38 Trennprinzip PFGE (>90°) Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.39 February 28, 2014 Pulsed Field Gel Electrophoresis 39 Trennprinzip PFGE (>90°) Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.40 February 28, 2014 Pulsed Field Gel Electrophoresis 40 Trennprinzip PFGE (>90°) Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.41 February 28, 2014 Pulsed Field Gel Electrophoresis 41 Trennprinzip PFGE (>90°) Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.42 February 28, 2014 Pulsed Field Gel Electrophoresis 42 Trennprinzip PFGE (>90°) Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.43 February 28, 2014 Pulsed Field Gel Electrophoresis 43 Trennprinzip PFGE (>90°) Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.44 February 28, 2014 Pulsed Field Gel Electrophoresis 44 Trennprinzip PFGE (>90°) Field Orientation
28. Februar 2014 Pulsfeldgelelektrophorese.45 February 28, 2014 Pulsed Field Gel Electrophoresis 45 Parameter Interval Voltage Agarose concentration Temperature Angle Duration Upper Limit of Separation distance changes by modified number of pulses Sharpness of bands Duration of Electrophoresis Distance of bands from starting point Distance between different sized bands
28. Februar 2014 Pulsfeldgelelektrophorese.46 February 28, 2014 Pulsed Field Gel Electrophoresis 46 Theory Bustamante, Tibtech, 1993 DNA of a T2 Phage (164kb) Conventional Electrophoresis visualized with a Fluorescence microskope 1% Agarose, 8V/cm (= 240V with Rotaphor)
28. Februar 2014 Pulsfeldgelelektrophorese.47 February 28, 2014 Pulsed Field Gel Electrophoresis 47 Trapping Molecules > 1000 kbp result increasingly in additional loops the larger the DNA the more loops the faster (high voltage) the stronger increases with higher agarose concentration handling limit 0,5% Yeast chromosome ~1Mbp direction Break through Blocking Spiral Dead end
28. Februar 2014 Pulsfeldgelelektrophorese.48 February 28, 2014 Pulsed Field Gel Electrophoresis 48 Trapping Example Duration 252h = 10,5 Tage Trapping inceases with voltage exponentially Separation of the smallest human Chromosome takes 1,5 years Homo sapiensS. pombeS. cerevisiae 5,7 Mbp 3,0 Mbp 1,5 Mbp
28. Februar 2014 Pulsfeldgelelektrophorese.49 February 28, 2014 Pulsed Field Gel Electrophoresis 49 Realization CHEF RGE TAFE RFE Biorad Biometra
28. Februar 2014 Pulsfeldgelelektrophorese.50 February 28, 2014 Pulsed Field Gel Electrophoresis 50 Rotaphor Electrodes
28. Februar 2014 Pulsfeldgelelektrophorese.51 February 28, 2014 Pulsed Field Gel Electrophoresis 51 Rotordesign Orientation of Pulse
28. Februar 2014 Pulsfeldgelelektrophorese.52 February 28, 2014 Pulsed Field Gel Electrophoresis 52 Comparison Size of gel: Slots Moving parts Buffer Refrigeration cascadable Very short pulses Dropout memory 20 x 20 cm 18 (– 50) yes, stepper motor Internal cooling Yes Not viable Month 13x14cm 15 No External cooling No Not usefull 3 h
28. Februar 2014 Pulsfeldgelelektrophorese.53 February 28, 2014 Pulsed Field Gel Electrophoresis 53 Salmonella Typing
28. Februar 2014 Pulsfeldgelelektrophorese.54 February 28, 2014 Pulsed Field Gel Electrophoresis 54 Salmonella Typing